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ItemIdentifying the function of milk protein genes and factors regulating their expression by exploiting the lactation strategy of the Tammar Wallaby (Macropus eugenii)(University of Melbourne, 2010)Milk is a crucial element for the development of the young, as well as mammary gland function. Unlike eutherian species, marsupials have a unique reproduction strategy, consisting of a short gestation, followed by a birth of an altricial young and a relatively long lactation. Additionally, marsupials progressively change the composition of their milk during lactation, exhibiting a phase-specific expression of whey milk proteins. This project exploited the lactation strategy of the tammar wallaby (Macropus eugenii) to analyse the function of whey acidic protein (WAP) in relation to mammary gland development. Furthermore, cross species promoter analysis together with microarray assay was employed to discover factors involved in the regulation of WAP, late lactation protein-A (LLP-A) and late lactation protein-B (LLP-B) gene expression. Structural and expression differences between marsupial and eutherian WAPs have presented challenges to identifying physiological functions of the WAP protein. This study characterized the genomic structure of tammar WAP (tWAP) gene and investigated the potential function of the protein. The in vitro studies demonstrated that tWAP and domain III (Dill) of the protein alone stimulate proliferation of a mouse mammary epithelial cell line (HC11) and primary cultures of tammar mammary epithelial cells (Wall-MEC), whereas deletion of Dill from tWAP abolishes this proliferative effect of the protein. Furthermore, the ability of Wall-MEC cells to form mammospheres was increased in the presence of tWAP. However, tWAP does not stimulate proliferation of human embryonic kidney (HEK293) or human gastric cancer (AGS) cells. DNA synthesis and expression of cyclin D1 and cyclin dependent kinase-4 (CDK-4) genes were significantly up-regulated when Wall-MEC and HC11 cells were grown in the presence of either tWAP or DHL These data suggest that Dill is the functional domain of the tWAP protein and that evolutionary pressure has led to the loss of this domain in eutherians, most likely as a consequence of adopting a reproductive strategy that relies on greater investment in development of the newborn during pregnancy. The temporal expression of marsupial milk protein genes such as WAP, LLP-A and LLP- 13 in marsupials, suggests that these genes are likely to be regulated by different molecular mechanisms from those milk protein genes expressed in eutherians. The in silico characterization of tammar wallaby, stripe-faced dunnart and short-tailed opossum WAP gene promoters identified putative recognition sequences for transcription factors such as STAT5, GR, NF-1, C/EBP, YY1 and SPI, which were conserved between the three marsupial species. Furthermore, WAP promoters demonstrated a prolactin- dependent transcriptional activity in vitro. However, although the analysis of LLP-A and LLP-B gene promoters from tammar wallaby, brush-tailed possum and short-tailed opossum revealed number of recognition sequences for transcription factors mentioned above, no transcriptional activity was observed regardless of the hormone combination used in the assay. This suggests that tammar wallaby WAP, LLP-A and LLP-B genes are regulated by different mechanisms during lactation. This thesis also for the first time describes the cloning, characterization and expression of marsupial specific LLP-A and LLP-B genes from fat-tailed dunnart and short-tailed opossum. Furthermore, microarray-based analysis of changes in gene expression between phase 2B and phase 3 of tammar wallaby lactation detected number of up-regulated genes during later stages of tammar wallaby lactation, which could be playing a role in the regulation of LLP-A and LLP-B gene expression. Overall, this project has highlighted the benefit of studying animals with extreme adaptation to lactation, such as the tammar wallaby, as a model to unravel the function of milk proteins in relation to mammary gland development and identify factors regulating their expression.